Biochemical characterization and process stability of polyphenoloxidase extracted from Victoria grape (Vitis vinifera ssp. Sativa)

Polyphenol oxidase (PPO) was isolated from Victoria grapes (Vitis vinifera ssp. Sativa) grown in South Africa and its biochemical characteristics were studied. Optimum pH and temperature for grape PPO activity were pH 5.0 and T = 25 °C with 10 mM catechol in McIlvaine buffer as substrate. PPO showed activity using the following substances: catechol, 4 methyl catechol, d, l-DOPA, (+) catechin and chlorogenic acid. K_m and V_max values were 52.6 ± 0.00436 mM and 653 ± 24.0 OD_400 nm/min in the case of 10 mM catechol as a substrate. Eight inhibitors were tested in this study and the most effective inhibitors were found to be ascorbic acid, l-cysteine and sodium metabisulfite. Kinetic studies showed that the thermal inactivation of Victoria grape PPO followed first-order kinetics, with an activation energy, E_a = 225 ± 13.5 of kJ/mol. Both in semipurified extract and in grape juice, PPO showed a pronounced high pressure stability.     

Inhibition kinetics of catechin and ferulic acid on polyphenoloxidase from cephalothorax of Pacific white shrimp (Litopenaeus vannamei)

Inhibition kinetics and mode of catechin and ferulic acid towards polyphenoloxidase (PPO) from cephalothorax of Pacific white shrimp were investigated. Catechin or ferulic acid inhibited quinone formation catalysed by PPO in a dose dependent manner. Catechin showed mixed type reversible inhibition with K_i value of 1.4 mM, whereas ferulic acid exhibited non-competitive reversible inhibition with K_i value of 37 mM. With increasing concentrations, both catechin and ferulic acid had higher copper (Cu²⁺) reduction and copper chelating capacity (P < 0.05). Catechin or ferulic acid could react with intermediated browning reaction products, thereby preventing dopachrome formation. Thus, catechin or ferulic acid could inhibit melanosis in Pacific white shrimp with different modes of inhibition towards PPO.     

Purification and some properties of polyphenoloxidase in eggplant (Solanum melongena)

Polyphenoloxidase (EC 1.10.3.1) in eggplant (Solatium melongena L) was purified by ammonium sulphate fractionation, DEAE-Cellulofine and DEAE-Toyopearl chromatography and Sephadex G-100 gel filtration. The enzyme was purified about 110-fold with a recovery of 5%. The purified enzyme more quickly oxidised chlorogenic acid (5-caffeoylquinic acid, IUPAC) than 10 other substrates used. The K_m value for the enzyme was found to be 0·50 mM with respect to chlorogenic acid; the optimum pH of the enzyme was about 4 with enzyme stability between pH 5 and 8. The enzyme was completely inactivated after heat treatment at 75°C for 30 min or 80°C for 5 min. Sodium metabisulphite, potassium cyanide and sodium fluoride markedly inhibited the enzyme activity.     

Steam blanching effect on polyphenoloxidase,peroxidase and colour of mango (Mangifera indica L.) slices

The heat stability of peroxidase (POD) and polyphenoloxidase (PPO) was investigated in mango (Mangifera indica L.) slices, and the relative colour was studied after different steam blanching times. There was complete inactivation after 5 min for POD and 7 min for PPO. Steam blanching of 3 min gave residual activity of 2.85% and 8.33% for PPO and POD, respectively, and when compared with samples blanched for 5 min had no effect on colour over 20 days of storage. Correlation was found between activities of PPO, POD and colour change over 20 days. After 7 min steam blanching the browning index was stable but less than at 3 and 5 min because non-enzymic browning had occurred. This research suggests that yellowness (b) and lightness (L) values contribute positively to the browning index (BI), compared to redness (a).     

Effect of ferulic acid on inhibition of polyphenoloxidase and quality changes of Pacific white shrimp (Litopenaeus vannamei) during iced storage

Effects of ferulic acid (FA) on polyphenoloxidase (PPO) and the quality changes of Pacific white shrimp (Litopenaeus vannamei) during iced storage for 10 days were investigated. Both FA and oxygenated FA (OFA) with different concentrations (0.1%, 0.5%, 1% and 2% (w/v)) showed PPO inhibitory activity in a dose dependent manner. FA was generally more effective in PPO inhibition than was OFA. Based on activity staining, white shrimp PPO with an apparent molecular weight of 210 kDa was inhibited by FA. When whole shrimps were treated with FA solution with concentrations of 1% or 2% and stored in ice for up to 10 days, the increase in psychrophilic and mesophilic bacterial count were retarded, in comparison with the control and those treated with 1.25% sodium metabisulphite (SMS). The coincidental lower rates of increase in pH and total volatile base content were obtained. Additionally, shrimps treated with 2% FA possessed the lowest peroxide value and thiobarbituric acid reactive substances (TBARS) value during the storage. After 10 days of storage, shrimps treated with 2% FA had the lower melanosis score and higher score for colour, flavour and overall likeness, compared with the control and SMS treated shrimps (P < 0.05).     

Antioxidant properties of different fractions of tubers from Pueraria tuberosa Linn

Pueraria tuberosa Linn. (PT), Leguminosae, is a perennial climber, growing throughout tropical parts of India. In the Ayurvedic system of medicine, it is used as a drug of choice to manage pain, inflammation and other related diseases. The antioxidant potency of P. tuberosa was investigated for the first time. Total antioxidant capacity was determined using an ABTS^∗+ assay. Lipid peroxidation was assessed in terms of thiobarbituric acid-reactive substances by using egg-yolk homogenates as lipid-rich media. Superoxide radical-scavenging was measured using riboflavin-light-nitro blue tetrazolium (NBT) assay. Hydroxyl radical trapping potential was determined by evaluating hydroxyl radical induced deoxyribose degradation using thiobarbituric acid method. In order to assess the metal chelation property, hydroxyl radical induced deoxyribose degradation was evaluated in the absence of ethylenediaminetetraacetic acid. Both hexane and methanol fractions inhibited lipid peroxidation and also chelated the iron, showing potent antioxidant property.     

Characterization of phenoloxidase activity of carapace and viscera from cephalothorax of Norway lobster (Nephrops norvegicus)

The characterization of phenoloxidase activity was performed in both carapace and viscera extracts of Norway lobster. Phenoloxidase activity rose with increasing temperature up to 60 °C. The carapace enzymatic extract showed the highest themostability, retaining about 80% of maximum activity at 45 °C and 40% at 65 °C. On the other hand, both enzymatic extracts showed a single peak activity at neutral-slightly alkaline pH and were quite resistant to inactivation at alkaline pH (pH > 8), but phenoloxidase activity became unstable at pH lower than 5.5. The enzymatic extract obtained from viscera showed a higher affinity for catechol (K_M 5.97 mM) than carapace extract (K_M 19.40 mM). Both mono- and diphenoloxidase activities were found in carapace and viscera. Polyphenoloxidase and converted-hemocyanin into PPO-like enzyme could be the main responsible for these activities.     

Evaluation of antioxidant and antimicrobial properties of finger millet polyphenols (Eleusine coracana)

Phenolic acids from finger millet (Eleusine coracana) milled fractions (whole flour, seed coat, 3%, 5% and 7%) were isolated and their antioxidant and antimicrobial properties were evaluated. Acidic methanol extracts from seed coat to whole flour were rich in polyphenol content and were found to be stable up to 48 h at pH 4, 7, and 9 as studied by ultraviolet spectroscopy. Diadzene, gallic, coumaric, syringic and vanillic acids were identified as major phenolic acids from the extracted phenolics. Diadzene content was highest in concentration in the 5% flour. The reducing power of seed coat extract was significantly (p < 0.05) higher than that of whole flour extract. Antioxidant activity (AA) as determined by the β-carotene–linoleic acid assay indicated that the AA was highest in seed coat extract (86%), whilst at the same concentration it was only 27% in the whole flour extract. The seed coat extract showed higher antimicrobial activity against Bacillus cereus and Aspergillus flavus compared to whole flour extract. From these observations, it can be inferred that the polyphenols are responsible for the microbial activity of the millet and the results indicate that potential exists to utilise finger millet seed coat as an alternative natural antioxidant and food preservative.     

荸荠多糖的提取及特性研究

采用超声波辅助法从荸荠中提取多糖,并研究其理化性质与组成。通过考察料液比、超声波辐射时间、温度以及超声波功率4个因素,设计正交试验,得出超声波辅助法提取荸荠多糖的最优工艺条件为:料液比1:12、超声波辐射时间为40min、超声波功率80W、温度为70℃,荸荠多糖纯度为78.8%。多糖理化特性和组分分析表明,荸荠多糖易溶于热水,不含淀粉、酚类物质,主要由葡萄糖和半乳糖组成,二者摩尔比为半乳糖:葡萄糖=1:10。     

A comparative analysis of property of lychee polyphenoloxidase using endogenous and exogenous substrates

Lychee polyphenoloxidase (PPO) was extracted and partially purified using ammonium sulphate precipitation and dialysis. The comparative analysis of PPO property was performed using its endogenous substrate (–)-epicatechin and exogenous substrate catechol. The pH optima for activity and activation temperature profiles of lychee PPO were very different when the enzyme reacted with endogenous and exogenous substrates. The addition of ethylenediaminetetraacetic acid disodium salt into the endogenous or exogenous substrate–enzyme system exhibited the same lowest inhibition of the PPO activity. However, l-cysteine was most effective in inhibiting enzymatic activity in the endogenous substrate–enzyme system while ascorbic acid was the best inhibitor in the exogenous substrate–enzyme system. Fe²⁺ greatly accelerated the enzymatic reaction between endogenous substrate and PPO, but Cu²⁺ exerted the same effect on the reaction between exogenous substrate and PPO. Based on the kinetic analysis, lychee PPO could strongly bind endogenous substrate but it possessed a higher catalytic efficiency to exogenous substrate.     

In vitro bioaccessibility,bioavailability and plasma protein interaction of polyphenols from Annurca apple (M. pumila Miller cv Annurca)

The in vitro bioaccessibility, bioavailability and plasma protein interaction of polyphenols from Annurca apple and other conventional cultivars were evaluated. Salivary digestion concentrated into the medium 27–35% of native apple polyphenols, suggesting the potential bioavailability through the oral mucosal epithelium of significant amounts of bioactive compounds that could be gastric sensitive and/or poorly absorbed in the intestine. Annurca flesh revealed the highest content and provided the best intestinal bioaccessibility and bioavailability of oligomeric procyanidins among all of the apple peel and flesh tested. Since 49.4% of native procyanidins were not absorbed, they are expected to accumulate in the intestinal lumen where a potential inhibition capacity of cellular cholesterol uptake could be assumed. The permeated procyanidins (6.7% of their native pattern, 12.0% of intestinal procyanidins) significantly bound (58.7%) to plasma HDLs, suggesting a major role in cholesterol metabolism. Our results would indicate Annurca apple and its potential nutraceuticals as effective in the regulation of plasma cholesterol levels.     

Biochemical properties of pepsinogen and pepsin from the stomach of albacore tuna (Thunnus alalunga)

Pepsinogen (PG) from the stomach of albacore tuna (Thunnus alalunga) was purified to homogeneity by using a series of chromatographies involving Sephacryl S-200HR, Sephadex G-50 and DEAE-cellulose with a 658-fold increase in purity. Based on the native-PAGE and zymography, PG showed a single band with pepsin activity. Molecular weights (MW) of PG and active pepsin were estimated to be 39.9 and 32.7 kDa as determined by SDS–PAGE, respectively. PG was converted to the corresponding pepsin through an intermediate form (MW ≈ 36.8 kDa) and the complete activation was observed after 30–60 min. The N-terminal amino acid sequence of the first 15 amino acids of activation segment of pepsinogen was FHKLPLIKGKTAREE. The optimal pH and temperature for pepsin activity were 2.0 and 50 °C, respectively. The activity was stable in the pH range of 2–5. Residual activity more than 85% was found after heating at temperatures up to 50 °C for 30 min. Pepsin activity was strongly inhibited by pepstatin A, whilst E-64, ethylenediaminetetraacetic acid (EDTA) and soybean trypsin inhibitor exhibited the negligible effect. SDS and cysteine also showed inhibitory effects, whilst ATP, molybdate, NaCl and CaCl₂ had no impact on pepsin activity.     

Free radical scavenging, inhibition of polyphenoloxidase activity and copper chelating properties of model Maillard systems

Antioxidant properties of Maillard reaction products (MRP) obtained from aqueous mixtures of glucose (0.8 mol/l) with proline, glycine, arginine, lysine, cysteine or glutathione (0.5 mol/l) heated at 103 °C for 1-92 h were investigated using different in vitro tests. Free radical scavenging activity was determined by measuring their reactivity towards DPPH° and lipidic radicals produced by AAPH. Contrary to glucose-proline and glucose-glycine MRP, glucose-lysine and glucose-arginine MRP displayed high scavenging activities. The activity of glucose-lysine MRP peaked after 14 h of heating while the activity of glucose-arginine constantly increased during the whole heat treatment. The high scavenging capacity observed towards DPPH° for glucose-cysteine mixtures could be attributed to the sulfhydryl group of cysteine.The inhibitory effect of MRP on activity of two copper-oxidoreductases, apple polyphenoloxidase and mushroom tyrosinase, assessed by polarography, showed that thiol-derived MRP were the most potent inhibitors, even at very low levels in the reaction medium. Conversely, the other MRP were only slightly efficient at high levels. Unheated mixtures containing thiol compounds exhibited a potent copper chelating ability, as efficient as EDTA, when determined by the tetramethylmurexide (TMM) test. After heating, these mixtures lost part of their chelating efficiency, but it remained higher than that of the other MRP, suggesting that the sulfhydryl group also played a role in the copper chelating properties.     

Evidence of an active laccase-like enzyme in deepwater pink shrimp (Parapenaeus longirostris)

This paper demonstrates the presence of an active laccase-like enzyme from deepwater pink shrimp (Parapenaeus longirostris) using polyacrylamide gel electrophoresis. This enzyme was found in all anatomical parts of the deepwater pink shrimp, but particularly in the cephalothorax, and became active during the course of storage. Gel staining with laccase-specific substrates such as ADA, DMP and DAB was used to characterize a protein of around 44 kDa as containing laccase activity. The enzyme was inhibited by a specific inhibitor, CTAB. 4-Hexylresorcinol, a specific inhibitor of polyphenoloxidase (PPO), did not inhibit the laccase-like enzyme. Low concentrations of antioxidants ascorbic acid or sodium metabisulphite were sufficient to inhibit the laccase-like enzyme. ABTS and DMP were subsequently used to characterize the enzyme. Given the evidence of this enzyme in deepwater pink shrimp, new melanosis-inhibiting compounds that are suitable for consumption need to be found to complement specific inhibitors of PPO activity.     

Effect of temporal variation,gender and size on cuticle polyphenol oxidase activity in deep-water rose shrimp (Parapenaeus longirostris)

Seasonal changes and physiological factors related to sex, size and spawning period may alter polyphenol oxidase (PPO) activity in crustacean decapods. Given the undesirable effects that the enzyme has on the marketability of shrimps, the cuticles of the valuable deep-water rose shrimp were examined. Monthly measurements of PPO activity in cuticles of juvenile and adult males and females were recorded for one year, along with sea-surface temperature (SST) and photoperiod. PPO activity was highest during late summer (August and September) and was lowest between February and March. The rate of enzyme activity in males was double that of females, when corrected for time period. Juvenile shrimp had the highest enzyme activity. Under the conditions tested, our results support that PPO assessment should take into account the sex and size as factors that potentially biasing in tissue enzyme distributions.     

Microencapsulation by spray drying of bioactive compounds from cactus pear (Opuntia ficus-indica)

Bioactive compounds of pulp (CP) and ethanolic (CE) extracts of the cactus pear (Opuntia ficus-indica) were encapsulated with maltodextrin (MD) or inulin (I). A 2² statistical factorial design was then used to study the stability of the powders obtained at the optimal conditions for each system (CP–MD, CP–I, CE–MD and CE–I) at 60 °C in the dark. The 3:1 ratio of core/coating material and 140 °C inlet air temperature were the optimal conditions for CP–MD and CE–MD systems; whereas, for CP–I and CE–I, the ratios were 3:1 and 5:1, respectively, and 120 °C was used for the inlet air temperature for both systems. An increase of phenolic compounds was observed in all systems during storage at 60 °C. Indicaxanthins in all systems showed a slow degradation during storage at 60 °C and were more stable than betacyanins. The microcapsules described in this study represent an interesting food additive for incorporation into functional foods, due to both the presence of antioxidants and as a red colourant.     

Nutritional constituents and antioxidant properties of indigenous kembayau (Dacryodes rostrata (Blume) H. J. Lam) fruits

The nutritional and antioxidant properties of peels, pulp and seeds of kembayau (Dacryodes rostrata) fruits were evaluated. Kembayau seeds and pulp were rich in fat, while peels had the highest ash contents. Potassium was the most prevalent mineral in peels (380.72-1112.00 mg/100 g). In kembayau fruits, total flavonoid content (1012.74-28,022.28 mg rutin equivalent/100 g) was higher than total phenolic and total monomeric anthocyanin contents. Kembayau seeds exhibited high flavonoid and phenolic contents compared to the contents in peels and pulp. Antioxidant capacities were also higher in seeds as typified by trolox equivalent antioxidant capacity assay (51.39-74.59 mmol TE/100 g), ferric reducing antioxidant power assay (530.05-556.98 mmol Fe²⁺/100 g) and by 1,1-diphenyl-2-picryl hydrazyl radical scavenging activity (92.18-92.19%) when compared to peels and pulp. Pulp and peels of kembayau fruit may be an important source of energy and minerals for human consumption, while seeds have a good potential as antioxidants.     

Polyphenolic composition in different parts of some cultivars of globe artichoke (Cynara cardunculus L. var. scolymus (L.) Fiori)

The analysis of polyphenols of leaves and different parts (outer, intermediate and inner bracts, and receptacle) of heads in five globe artichoke cultivars of Campania region (Italy) and one accession of cultivated cardoon was performed. Data obtained suggest that the edible parts (receptacles with inner and intermediate bracts) of these cultivars of artichoke could represent a good source of health-promoting polyphenols and therefore encourage a nutriceutical use of this species, as an alternative to the more traditional phytopharmaceutical applications of leaf extracts. Moreover, it was demonstrated that single polyphenols accumulate preferentially in specific parts of the heads and in specific genotypes.     

Volatile compounds and descriptive odor attributes in umbu (Spondias tuberosa) fruits during maturation

The umbu fruit is appreciated in north and northeast region of Brazil mainly because of its refreshing and acidic flavor. The objective of this work was to determine the volatile compounds in half-ripe and ripe stages of umbu fruit and to identify important characteristic compounds which could contribute for its aroma by performing sniffing evaluation of volatile extracts. The principal volatile compounds identified in the ripe umbu fruit pulp were 4-methyl-3-penten-2-one, ethyl benzene, 1-penten-3-one, 2-acetyl thiazone, p-xylene, limonene, 2,2-dimethyl 4-octenal, 3-hexanol, 2-nonanol, 1-nonanol, 2-pentanol, 2-octanol, 3-methylethyl 2-butanoate, butyl benzoate, 3-allyl cyclohexene, 2-acetyl furan, 2-hexyl furan, ??-caryophyllene and methyl pyrazine. A total of 37 volatile compounds were diagnosed with characteristic aroma attributes. The principal volatile compounds which could be responsible for the characteristic aroma of ripe umbu fruit pulp were ??-(Z)-ocimene, methyl pyrazine, 2-butyl-thiophene, methyl octanoate, 2-hexyl furan, 2-octanol, (E)-2-cyclohexen-1-one, 3-bromo-cyclohexene, 1-heptanol, 2-nonanol and 1-octanol.